Photocytotoxicity of copper(II) complexes of curcumin and N-ferrocenylmethyl-L-amino acids

Copper(II) complexes Cu(Fc-aa)(cur)] (1-3) of curcumin (Hcur) and N-ferrocenylmethyl-L-amino acids (Fc-aa), viz., ferrocenylmethyl-L-tyrosine (Fc-TyrH), ferrocenylmethyl-L-tryptophan (Fc-TrpH) and ferrocenylmethyl-L-methionine (Fc-MetH), were prepared and characterized. The DNA photocleavage activity, photocytotoxicity and cellular localization in HeLa and MCF-7 cancer cells of these complexes were studied. Acetylacetonate (acac) complexes Cu(Fc-aa)(acac)] (4-6) were prepared and used as controls. The chemical nuclease inactive complexes showed efficient pUC19 DNA cleavage activity in visible light. Complexes 1-3 showed high photocytotoxicity with low dark toxicity thus giving remarkable photodynamic effect. FACScan analysis showed apoptosis of the cancer cells. Fluorescence microscopic studies revealed primarily cytosolic localization of the complexes. (C) 2013 Elsevier Masson SAS. All rights reserved.

Osteoprotective effect of propranolol in ovariectomized rats: a comparison with zoledronic acid and alfacalcidol

Currently beta-adrenergic receptor blockers are considered to be potential drugs under investigation for preventive or therapeutic effect in osteoporosis. However, there is no published data showing the comparative study of beta-blockers with well accepted agents for the treatment of osteoporosis. To address this question, we compared the effects of propranolol with well accepted treatments like zoledronic acid and alfacalcidol in an animal model of postmenopausal osteoporosis. Five days after ovariectomy, 36 ovariectomized (OVX) rats were divided into 6 equal groups, randomized to treatments zoledronic acid (100 mu g/kg, intravenous single dose); alfacalcidol (0.5 mu g/kg, oral gauge daily); propranolol (0.1 mg/kg, subcutaneously 5 days per week) for 12 weeks. Untreated OVX and sham OVX were used as controls. At the end of treatment serum calcium and alkaline phosphatase were assayed. Femurs were removed and tested for bone density, bone porosity, bone mechanical properties and trabecular micro-architecture. Propranolol showed a significant decrease in alkaline phosphatase levels and bone porosity in comparison to OVX control. Moreover, propranolol significantly improved bone density, bone mechanical properties and inhibited the deterioration of trabecular microarchitecture when compared with OVX control. The osteoprotective effect of propranolol was comparable with zoledronic acid and alfacalcidol. Based on this comparative study, the results strongly suggest that propranolol can be a candidate therapeutic drug for the management of postmenopausal osteoporosis.

Methionine down-regulates TLR4/MyD88/NF-kappa B signalling in osteoclast precursors to reduce bone loss during osteoporosis

Background and PurposeStudies have demonstrated that a moderate intake of amino acids is associated with development of bone health. Methionine, a sulphur-containing essential amino acid, has been largely implicated for improving cartilage formation, however its physiological significance on bone integrity and functionality have not been elucidated. We investigated whether methionine can prevent osteoporotic bone loss. Experimental ApproachThe anti-resorptive effect of methionine, (250mgkg(-1) body wt administered in drinking water for 10 weeks), was evaluated in ovariectomized (OVX) rats by monitoring changes in bone turnover, formation of osteoclasts from blood-derived mononuclear cells and changes in the synthesis of pro-osteoclastogenic cytokines. Key resultsMethionine improved bone density and significantly decreased the degree of osteoclast development from blood mononuclear cells in OVX rats, as indicated by decreased production of osteoclast markers tartarate resistant acid phosphatase b (TRAP5b) and MIP-1. siRNA-mediated knockdown of myeloid differentiation primary response 88 MyD88], a signalling molecule in the toll-like receptor (TLR) signalling cascade, abolished the synthesis of both TRAP5b and MIP-1 in developing osteoclasts. Methionine supplementation disrupted osteoclast development by inhibiting TLR-4/MyD88/NF-B pathway. Conclusions and ImplicationsTLR-4/MyD88/NF-B signalling pathway is integral for osteoclast development and this is down-regulated in osteoporotic system on methionine treatment. Methionine treatment could be beneficial for the treatment of postmenopausal osteoporosis.

Selective inhibition of IFNG-induced autophagy by Mir155- and Mir31-responsive WNT5A and SHH signaling

Autophagy is one of the major immune mechanisms engaged to clear intracellular infectious agents. However, several pathogens have evolved strategies to evade autophagy. Here, we demonstrated that Mycobacteria, Shigella, and Listeria but not Klebsiella, Staphylococcus, and Escherichia inhibit IFNG-induced autophagy in macrophages by evoking selective and robust activation of WNT and SHH pathways via MTOR. Utilization of gain- or loss-of-function analyses as well as mir155-null macrophages emphasized the role of MTOR-responsive epigenetic modifications in the induction of Mir155 and Mir31. Importantly, cellular levels of PP2A, a phosphatase, were regulated by Mir155 and Mir31 to fine-tune autophagy. Diminished expression of PP2A led to inhibition of GSK3B, thus facilitating the prolonged activation of WNT and SHH signaling pathways. Sustained WNT and SHH signaling effectuated the expression of anti-inflammatory lipoxygenases, which in tandem inhibited IFNG-induced JAK-STAT signaling and contributed to evasion of autophagy. Altogether, these results established a role for new host factors and inhibitory mechanisms employed by the pathogens to limit autophagy, which could be targeted for therapeutic interventions.

Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16

We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsinf-old and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. (C) 2013 The Authors. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.

Ferrocenyl-L-amino acid copper(II) complexes showing remarkable photo-induced anticancer activity in visible light

Ferrocene-conjugated copper(II) complexes Cu(Fc-aa)(aip)](ClO4) (1-3) and (Cu(Fc-aa)(pyip)](ClO4) (4-6) of L-amino acid reduced Schiff bases (Fc-aa), 2-(9-anthryl)-1H-imidazo4,5-f]1,10]phenanthroline (aip) and 2-(1-pyrenyl)-1H-imidazo4,5-f] 1,10]phenanthroline (pyip), where Fc-aa is ferrocenylmethyl-L-tyrosine (Fc-Tyr in 1, 4), ferrocenylmethyl-L-tryptophan (Fc-Trp in 2, 5) and ferrocenylmethyl-L-methionine (Fc-Met in 3, 6), were prepared and characterized, and their photocytotoxicity was studied (Fc = ferrocenyl moiety). Phenyl analogues, viz. (Cu(Ph-Met)(aip)](ClO4) (7) and (Cu(Ph-Met)(pyip)](ClO4) (8), were prepared and used as control compounds. The bis-imidazophenanthroline copper(II) complexes, viz. (Cu(aip)(2)(NO3)](NO3) (9) and Cu(pyip)(2)(NO3)](NO3) (10), were also prepared and used as controls. Complexes 1-6 having a redox inactive cooper(II) center showed the Fc(+)-Fc redox couple at similar to 0.5 V vs. SCE in DMF-0.1 mol (Bu4N)-N-n](ClO4). The copper(II)-based d-d band was observed near 600 nm in DMF-Tris-HCl buffer (1 :1 v/v). The ferrocenyl complexes showed low dark toxicity, but remarkably high photocytotoxicity in human cervical HeLa and human breast adenocarcinoma MCF-7 cancer cells giving an excellent photo-dynamic effect while their phenyl analogues were inactive. The photo-exposure caused significant morphological changes in the cancer cells when compared to the non-irradiated ones. The photophysical processes were rationalized from the theoretical studies. Fluorescence microscopic images showed 3 and 6 localizing predominantly in the endoplasmic reticulum (ER) of the cancer cells, thus minimizing any undesirable effects involving nuclear DNA.

Zoledronic acid in combination with alfacalcidol has additive effects on trabecular microarchitecture and mechanical properties in osteopenic ovariectomized rats

We conducted the present study to investigate the therapeutic effects of the antiresorptive agent zoledronic acid (ZOL), alone and in combination with alfacalcidol (ALF), in a rat model of postmenopausal osteoporosis. Female Wistar rats were ovariectomized (OVX) or sham-operated at 3 months of age. Twelve weeks post surgery, rats were randomized into six groups: (1) sham + vehicle, (2) OVX + vehicle, (3) OVX + ZOL (100 mu g/kg, i.v. single dose), (4) OVX + ZOL (50 mu g/kg, i.v. single dose), (5) OVX + ALF (0.5 mu g/kg, oral gauge daily) and (6) OVX + ZOL (50 mu g/kg, i.v. single dose) + ALF (0.5 mu g/kg, oral gauge daily) for 12 weeks. After treatment, we evaluated the mechanical properties of the lumbar vertebra and femoral mid-shaft. Femurs were also tested for bone density, porosity and trabecular micro-architecture. Biochemical markers in serum and urine were also determined. With respect to improvement in the mechanical strength of the lumbar spine and the femoral mid-shaft, the combination treatment of ZOL and ALF was more effective than each administered as a monotherapy. Moreover, combination therapy using ZOL and ALF preserved the trabecular micro-architecture and cortical bone porosity. Furthermore, the combination treatment of ZOL and ALF corrected the decrease in serum calcium and increase in serum alkaline phosphatase and the tartarate-resistant acid phosphatase level better than single-drug therapy using ZOL or ALF in OVX rats. In addition, the combination treatment of ZOL and ALF corrected the increase in urine calcium, phosphorous and creatinine levels better than single-drug therapy using ZOL or ALF in OVX rats. These data suggest that the combination treatment of ZOL and ALF has a therapeutic advantage over each monotherapy for the treatment of osteoporosis.

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of beta-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.

Development, in vitro and in vivo characterization of zoledronic acid functionalized hydroxyapatite nanoparticle based formulation for treatment of osteoporosis in animal model

We investigated the potential of using novel zoledronic acid (ZOL)-hydroxyapatite (HA) nanoparticle based drug formulation in a rat model of postmenopausal osteoporosis. By a classical adsorption method, nanoparticles of HA loaded with ZOL (HNLZ) drug formulation with a size range of 100-130 nm were prepared. 56 female Wistar rats were ovariectomized (OVX) or sham-operated at 3 months of age. Twelve weeks post surgery, rats were randomized into seven groups and treated with various doses of HNLZ (100, 50 and 25 mu g/kg, intravenous single dose), ZOL (100 mu g/kg, intravenous single dose) and HA nanoparticle (100 mu g/kg, intravenous single dose). Untreated OVX and sham OVX served as controls. After three months treatment period, we evaluated the mechanical properties of the lumbar vertebra and femoral mid-shaft. Femurs were also tested for trabecular microarchitecture. Sensitive biochemical markers of bone formation and bone resorption in serum were also determined. With respect to improvement in the mechanical strength of the lumbar spine and the femoral mid-shaft, the therapy with HNLZ drug formulation was more effective than ZOL therapy in OVX rats. Moreover, HNLZ drug therapy preserved the trabecular microarchitecture better than ZOL therapy in OVX rats. Furthermore, the HNLZ drug formulation corrected increase in serum levels of bone-specific alkaline phosphatase, procollagen type I N-terminal propeptide, osteocalcin, tartrate-resistant acid phosphatase 5b and C-telopeptide of type 1 collagen better than ZOL therapy in OVX rats. The results strongly suggest that HNLZ novel drug formulation appears to be more effective approach for treating severe osteoporosis in humans. (C) 2014 Elsevier B.V. All rights reserved.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, Groundnut bud necrosis virus

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 50 a phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

The peptide NCbz-Val-Tyr-OMe and aromatic pi-pi interactions

The peptide N-benzyloxycarbonyl-L-valyl-L-tyrosine methyl ester or NCbz-Val-Tyr-OMe (where NCbz is N-benzyloxycarbonyl and OMe indicates the methyl ester), C23H28N2O6, has an extended backbone conformation. The aromatic rings of the Tyr residue and the NCbz group are involved in various attractive intra- and intermolecular aromatic - interactions which stabilize the conformation and packing in the crystal structure, in addition to NH...O and OH...O hydrogen bonds. The aromatic - interactions include parallel-displaced, perpendicular T-shaped, perpendicular L-shaped and inclined orientations.

Insulin-Like Growth Factor System in Cancer: Novel Targeted Therapies

Insulin-like growth factors (IGFs) are essential for growth and survival that suppress apoptosis and promote cell cycle progression, angiogenesis, and metastatic activities in various cancers. The IGFs actions are mediated through the IGF-1 receptor that is involved in cell transformation induced by tumour. These effects depend on the bioavailability of IGFs, which is regulated by IGF binding proteins (IGFBPs). We describe here the role of the IGF system in cancer, proposing new strategies targeting this system. We have attempted to expand the general viewpoint on IGF-1R, its inhibitors, potential limitations of IGF-1R, antibodies and tyrosine kinase inhibitors, and IGFBP actions. This review discusses the emerging view that blocking IGF via IGFBP is a better option than blocking IGF receptors. This can lead to the development of novel cancer therapies.

Insights into stabilizing interactions in the distorted domain-swapped dimer of Salmonella typhimurium survival protein

The survival protein SurE from Salmonella typhimurium (StSurE) is a dimeric protein that functions as a phosphatase. SurE dimers are formed by the swapping of a loop with a pair of beta-strands and a C-terminal helix between two protomers. In a previous study, the Asp230 and His234 residues were mutated to Ala to abolish a hydrogen bond that was thought to be crucial for C-terminal helix swapping. These mutations led to functionally inactive and distorted dimers in which the two protomers were related by a rotation of 167 degrees. New salt bridges involving Glu112 were observed in the dimeric interface of the H234A and D230A/H234A mutants. To explore the role of these salt bridges in the stability of the distorted structure, E112A, E112A/D230A, E112A/H234A, E112A/D230A/H234A, R179L/H180A/H234A and E112A/R179L/H180A/H234A mutants were constructed. X-ray crystal structures of the E112A, E112A/H234A and E112A/D230A mutants could be determined. The dimeric structures of the E112A and E112A/H234A mutants were similar to that of native SurE, while the E112A/D230A mutant had a residual rotation of 11 degrees between the B chains upon superposition of the A chains of the mutant and native dimers. The native dimeric structure was nearly restored in the E112A/H234A mutant, suggesting that the new salt bridge observed in the H234A and D230A/H234A mutants was indeed responsible for the stability of their distorted structures. Catalytic activity was also restored in these mutants, implying that appropriate dimeric organization is necessary for the activity of SurE.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.